title: A novel intermediate in the reaction of seleno CYP119 with m-chloroperbenzoic acid.
abstract: Cytochrome P450-mediated monooxygenation generally proceeds via a reactive ferryl intermediate coupled to a ligand radical [Fe(IV)═O]+• termed Compound I (Cpd I). The proximal cysteine thiolate ligand is a critical determinant of the spectral and catalytic properties of P450 enzymes. To explore the effect of an increased level of donation of electrons by the proximal ligand in the P450 catalytic cycle, we recently reported successful incorporation of SeCys into the active site of CYP119, a thermophilic cytochrome P450. Here we report relevant physical properties of SeCYP119 and a detailed analysis of the reaction of SeCYP119 with m-chloroperbenzoic acid. Our results indicate that the selenolate anion reduces rather than stabilizes Cpd I and also protects the heme from oxidative destruction, leading to the generation of a new stable species with an absorbance maximum at 406 nm. This stable intermediate can be returned to the normal ferric state by reducing agents and thiols, in agreement with oxidative modification of the selenolate ligand itself. Thus, in the seleno protein, the oxidative damage shifts from the heme to the proximal ligand, presumably because (a) an increased level of donation of electrons more efficiently quenches reactive species such as Cpd I and (b) the protection of the thiolate ligand provided by the protein active site structure is insufficient to shield the more oxidizable selenolate ligand.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517, United States.
authors.forename: Santhosh
authors.initials: S
authors.lastname: Sivaramakrishnan
authors.forename: Hugues
authors.initials: H
authors.lastname: Ouellet
authors.forename: Jing
authors.initials: J
authors.lastname: Du
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D002723:Chlorobenzoates / Q000737:chemistry* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D017279:Selenocysteine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry
chemical_list: D019843:Archaeal Proteins; D002723:Chlorobenzoates; D005290:Ferric Compounds; D005296:Ferrous Compounds; D017279:Selenocysteine; D003577:Cytochrome P-450 Enzyme System; C408874:CYP119 protein, Sulfolobus solfataricus; C000433:3-chloroperbenzoic acid